Beta-hexosyl-transferases and uses thereof

Abstract

This invention relates generally to the discovery of novel recombinant forms of β-hexosyl-transferases (BHT) and uses thereof to produce galacto-ligosaccharides (GOS) or as food additives.

What is claimed is: 
     
       1. A method for producing enzymatically active recombinant β-hexosyl-transferase (rBHT) protein which comprises
 transforming a  Pichia pastoris  cell with a plasmid under the control of a suitable promotor wherein the plasmid contains an isolated DNA encoding a rBHT protein having the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18 or 20, 
 
       wherein the promoter is operably linked to the isolated DNA, and
 culturing the transformed cell under conditions such that the rBHT protein is produced, 
 wherein the rBHT protein produced by the transformed cell has β-hexosyl-transferase enzymatic activity. 
 
     
     
       2. The method of  claim 1 , wherein the rBHT protein comprises the amino acid sequence of SEQ ID NO: 12 or 14. 
     
     
       3. The method of  claim 1 , wherein the nucleotide sequence of the isolated DNA has at least 97% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17 or 19. 
     
     
       4. The method of  claim 3 , wherein the nucleotide sequence of the isolated DNA has at least 97% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 11, 13, or 15. 
     
     
       5. The method of  claim 1 , wherein the isolated DNA is linked to a DNA encoding a signal peptide. 
     
     
       6. The method of  claim 5 , wherein the signal peptide is an  S. cerevisiae  α-factor signal peptide. 
     
     
       7. The method of  claim 1 , wherein the suitable promotor is an alcohol oxidase promotor. 
     
     
       8. The method of  claim 1 , wherein the enzymatically active rBHT protein has a specific activity of 8 U/mg at 20° C. 
     
     
       9. The method of  claim 1 , wherein the nucleotide sequence of the isolated DNA has at least 99.5% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17 or 19. 
     
     
       10. The method of  claim 9 , wherein the nucleotide sequence of the isolated DNA has at least 99.5% sequence identity with the nucleic acid sequence set forth in SEQ NO: 11, 13 or 15.